Safe and effective interferon-beta gene therapy for the treatment of multiple sclerosis by regulating biological activity through the design of interferon-beta-galectin-9 fusion proteins.

Multiple sclerosis (MS) is the most common demyelinating disease. Despite the historical use of interferon-beta (IFN-ß) for the treatment of patients with MS, concerns exist regarding the side effects of IFN-ß. In this study, we designed a series of novel IFN-ß fusion proteins containing galectin-9 (gal-9), which exerts immunosuppressive effects through the binding to its receptor on activated Th1 cells. We hypothesized that these fusion proteins would improve the therapeutic effects and reduce the side effects of IFN-ß. The IFN-ß-gal-9 fusion proteins showed less IFN-ß biological activity on non-T cells than IFN-ß alone. In vitro experiments using re-stimulated T cells isolated from mice with experimental autoimmune encephalomyelitis (EAE) showed that the IFN-ß-gal-9 fusion proteins suppressed activated T cells more effectively than IFN-ß. Moreover, in our in vivo experiments, the gene transfer of IFN-ß-gal-9 fusion protein-expressing plasmid DNA into EAE mice showed beneficial therapeutic effects without cytopenia, a known side effect of IFN-ß. In contrast, the gene transfer of IFN-ß-expressing plasmid DNA induced a rapid decrease in the white blood cell count, despite its therapeutic effect. These results indicate that gene therapy using IFN-ß-gal-9 fusion proteins is expected to be safe and effective for the treatment of MS.

Evaluation of antiviral effect of type I, II, and III interferons on direct-acting antiviral-resistant hepatitis C virus.

Treatment of hepatitis C virus (HCV) infection has greatly improved in the last 5 years because of the identification of direct-acting antivirals (DAAs). However, concerns exist regarding the emergence of drug resistance-associated substitutions (RASs). In this study, we evaluated the in vivo antiviral effect of three classes of interferons (IFNs), namely, types I, II, and III IFNs, on DAA-resistant HCVs. IFN-α2, IFN-γ, and IFN-λ1 were selected as typical types I, II, and III IFNs, respectively. Human hepatocyte-transplanted chimeric mice were infected with NS3-D168, NS5A-L31-, and NS5A-Y93-mutated HCVs, and the antiviral effect of IFN-α2, IFN-γ, and IFN-λ1 on these HCV RASs was examined. Chimeric mice infected with NS3- and NS5A-mutated HCVs were hydrodynamically injected with IFN-expressing plasmids to evaluate the antiviral effect of IFNs. Serum concentrations of IFNs were maintained for at least 42 days. We found that serum HCV level significantly decreased and serum and hepatic HCV levels reached below detection limit in 5/5 and 3/5 chimeric mice injected with IFN-γ- and IFN-λ1-expressing plasmids, respectively. The antiviral effect of IFN-α2 on DAA-resistant HCVs was weaker than that of IFN-γ and IFN-λ1. Serum ALT levels showed a small and transient increase in mice injected with the IFN-γ-expressing plasmid but not in mice injected with the IFN-λ1-expressing plasmid. However, no apparent histological damage was observed in the liver sections of mice injected with the IFN-γ-expressing plasmid. These results indicate that IFN-γ and IFN-λ1 are an attractive therapeutic option for treating infection caused by NS3- and NS5A-mutated HCV.

Amelioration of Experimental Autoimmune Encephalomyelitis in Mice by Interferon-Beta Gene Therapy, Using a Long-Term Expression Plasmid Vector.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Repeated injections of the interferon-ß (IFN-ß) protein are required for relapse prevention therapy in patients with MS. IFN-ß gene transfer can be an alternative treatment that continuously supplies IFN-ß protein to the patient without requiring repeated injections. In a previous study, we constructed a novel long-term IFN-ß-expressing plasmid vector (pMx-IFN-ß). In the present study, we examined whether gene transfer of pMx-IFN-ß could be effective for the treatment of MS in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Seven days after injection of the EAE-inducing peptide, the EAE mice received hydrodynamic injections pMx-IFN-ß. The severity of EAE symptoms in the pMx-IFN-ß-treated mice was significantly lower for 1 month than that observed in the untreated mice. An evaluation of blood-brain barrier (BBB) function, using Evans Blue, showed that injection of pMx-IFN-ß suppressed the BBB disruptions normally observed in EAE mice, while BBB disruptions remained evident in the untreated EAE mice. Histological analysis showed fewer invasive inflammatory cells in the spinal cords of the pMx-IFN-ß-treated mice than in the spinal cords of the other mice. Serum interferon gamma protein (IFN-γ) concentrations in the pMx-IFN-ß-treated mice were significantly lower than that in the untreated mice, indicating that IFN-ß gene transfer suppressed the production of IFN-γ from pathogenic T cells. These results indicate that IFN-ß transgene expression by single administration of the pMx-IFN-ß can be an effective long-term treatment for MS.

Targeted Delivery of Interferon Gamma Using a Recombinant Fusion Protein of a Fibrin Clot-Binding Peptide With Interferon Gamma for Cancer Gene Therapy.

Accelerated formation of fibrin clots in a tumor microenvironment can be used for targeted delivery of interferon gamma (IFNγ) to tumor cells. Here, we selected cysteine-arginine-glutamic acid-lysine-alanine (CREKA) as the fibrin clot-binding peptide and designed 2 types of fusion proteins for tumor targeting. The CREKA peptide was fused to IFNγ's C-terminus, with or without a matrix metalloproteinase-2 (MMP2)-cleavable linker (IFNγ-mmp-CREKA or IFNγ-CREKA, respectively). The former was designed to release IFNγ from IFNγ-mmp-CREKA bound to fibrin clots, to ensure IFNγ's function in the tumor milieu. IFNγ-activated sequence-dependent reporter gene expression in B16-BL6 cells revealed that the biological activities of IFNγ-CREKA and IFNγ were comparable, whereas that of IFNγ-mmp-CREKA was approximately 60% that of IFNγ. Plasma concentrations of IFNγ-CREKA and IFNγ-mmp-CREKA remained at effective levels for at least 4 weeks after gene transfer into mice. After gene transfer to tumor-bearing mice, intratumoral concentration of IFNγ in pCpG-IFNγ-mmp-CREKA group was tended to be higher than those of the other groups. Inhibition of colon-26 tumor growth was significantly more with gene transfer of IFNγ-mmp-CREKA than with IFNγ or IFNγ-CREKA. These results indicate that targeted delivery of IFNγ to fibrin clots through IFNγ-mmp-CREKA fusion can enhance the therapeutic efficacy of IFNγ in cancer gene therapy.

Interferon-Inducible Mx Promoter-Driven, Long-Term Transgene Expression System of Interferon-ß for Cancer Gene Therapy.

Gene therapy techniques aiming to induce the long-term interferon-ß (IFN-ß) expression are desirable for inhibiting cancer growth. However, there has been no success in this regard because IFN-ß significantly inhibits transgene expression. This study used the IFN-inducible Mx promoter to promote IFN-ß expression. The pMx-IFN-ß plasmid was constructed to achieve long-term IFN-ß expression. In cultured cells transfected with the Mx promoter-driven reporter protein plasmid, IFN-ß induced concentration-dependent expression of the reporter protein. After the hydrodynamic injection of pMx-IFN-ß into mice, the serum concentration of IFN-ß was maintained at ≥100 pg/mL for >1 month. IFN-ß expression was significantly suppressed by the co-injection of small interfering RNA targeting the interferon-α/ß receptor (IFNAR), suggesting that IFN-ß binding to IFNAR increased IFN-ß expression. Moreover, the hydrodynamic injection of pMx-IFN-ß significantly suppressed the growth of colon26 tumors in mice. In contrast, a conventional promoter-driven plasmid was less effective than pMx-IFN-ß in all the experiments. Taken together, these results indicate that the interferon-inducible Mx promoter-driven expression system effectively achieves long-term expression of IFN-ß and represents a potential tool for cancer gene therapy.